Immunosuppressive agents are currently employed in a wide variety of medical applications including clinical transplanatation, treatment of autoimmune diseases and investigations into basic immunological functions. In particulr, transplantation of organs represents the major solution to human organ failure but immunosuppression of the recipient is essential for graft survival in mismatched donor/recipient combinations.
The current understanding of graft rejection suggests that passenger antigen-presenting cells of donor type are a preprequisite for the induction of the immune response in the recipient which will eventually lead to the rejection of the animal graft. It has been shown that long-term in vitro culture of donor organs (several weeks under high oxygen tension) does selectively deplete such cells and allows successful transplantation.
Unfortunately, most of the drugs commonly used for immunosuppression are themselves toxic and must be administered systemically causing impairment of the host's immune system and other deleterious effects.
Research has established that a fungal product, cyclosporin, can be employed in clinical transplantation with some success. However, nephrotoxicity, hepatotoxicity, systemic administration leading to patient susceptibility to opportunistic infection, spontaneous lymphomas, and expensive long-term therapy are among the major drawbacks when using this product. From this earlier research, it is apparent that there still exists a demand for better and less toxic immunomodulating agents.
It is well established (for review see Taylor, A. 1971. The toxicology of sporidesmins and other epipolythiadioxopiperazines. In Microbial Toxins VII, pp 337-376. Edited by S. Kadis, A. Ciegler and S. J. Ajl. New York: Academic Press) that a variety of fungi produce epipolythiodioxopiperazines when cultured in a nutrient broth. These compounds have been investigated in vitro for their potential antibacterial, antifungal, anit-viral and amoebicidal activities. However, their application in vivo has been greatly curtailed by high cellular toxicity in mammals. Indeed, the same has been shown of Trown's (Trown, A. W. 1968. Biochem. Biophys. Res. Commun. 33, 402) synthesized model compound, 1,4-dimethyl-3,6-epidithia-2,5-dioxopiperazine. Accordingly, little is known regarding the effects of any of these fungal or synthesized compounds in vivo.
Experimental allergic encephalomyelitis is an autoimmune demylinating disease of the central nervous system, considered at present the optimal laboratory model for multiple sclerosis. Studies on the pathogenesis of this model disease strongly point to the cellular nature of the immune damage and it is thought that macrophages are important in both afferent and efferent limbs of the immune response leading to the pathological state of experimental allergic encephalomyelitis. Again, nothing is known concerning the effects of these fungal and/or synthesized compounds, belonging to the epipolythiodioxopiperazines, on the etiology of this disease.
One object of the present invention is to provide a method of treating biological material in order to prevent graft rejection in mismatched donor/recipient combinations. By the expression "biological material" is meant any material which may be involved in transplantation procedures such as, for example, single cells, clumps of cells, complete organs, groups of organs or any combination thereof, whether of donor or recipient origin.
A further object of the invention is to provide a method for treating autoimmune diseases.
According to the present invention, a method of treating biological material as defined, comprises subjecting the material with a compound of the general formula (1). ##STR4## alone or in association with one of more pharmaceutically acceptable carriers or diluents wherein:
R.sup.o and R.sup.1 are radicals selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy and acyloxy; R.sup.2 and R.sup.3 are radicals separately selected from the group consisting of hydrogen and alkyl; or together, represent a radical of the general formula (2): ##STR5## wherein R.sup.5, R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are radicals separately selected from the group consisting of hydrogen, alkyl, hydroxy, alkoxy, sulfate, and halogen; or R.sup.2 and R.sup.3 together, represent a radical of the general formula (3): ##STR6## wherein R.sup.10 and R.sup.11 both represent hydrogen; or together, represent a valence bond; R.sup.12, R.sup.13, R.sup.14, R.sup.15 are radicals selected from the group consisting of hydrogen, hydroxy, alkoxy, sulfate and acyloxy; and n is an integer selected from the range 2 to 4.
It has been found that the class of compounds known as epipolythiodioxopiperazines, according to Formula (1) exhibits anti-phagocytic and immunomodulating properties.
A number of compounds falling within the class of epipolythiodioxopiperazines have been found to be particularly effective in preventing graft rejection, in particular gliotoxin, gliotoxin-tri-sulfide, gliotoxin-tetra-sulfide, sporidesmin, 1,4-dimethyl-3,6-epidithio-2,5-dioxopiperazine and dehydrogliotoxin.
An advantage of the present invention is that it permits the treatment, not necessarily of the recipient, but rather of the donated biological material, for example an organ, in transplantation procedures.
One advantage of treating donated material is that it eliminates the side effects of the currently used immunosuppressive drugs. Specific side effects adverted by the treatments of the present invention include toxicity to organs in the recipient, development of opportunistic infections such as pneumonia, expansive long-term therapy, etc.
It is hypothesised that the treatment of donor material according to the invention results in the selective inactivation of passenger leucocytes within the donor material which are responsible for initiation of graft rejection within the recipient. The treatments according to the invention, unlike conventional treatments, irreversibly inactivate the passenger leucocytes, thereby avoiding the need for long-term therapy of the recipient and the resulting deleterious side effects.
It is known that the species of the fungi Aspergillus and Penicillium, and other related fungi, generate metabolites in in vitro cultures that belong the epipolythiodioxopiperazine class of compounds, of which gliotoxin is one, and which can be obtained by modifications of well-published methods (eg. Lowe, G., et al. 1966. J. Chem. Soc., 1799, Dingley et al. 1962. J. Gen. Microbiol., 29, 127.).
We have now established that these compounds, display anti-phgocytic activity as tested by macrophage adherence to plastic as well as phagocytosis of particulate matter and, when used to pretreat stimulator spleen cells, inhibit the cells' ability to induce alloreactive and major histocompatibility complex restricted cytotoxic T cells. This is the model system for graft rejection. The metabolites (see FIG. 1) were chloroform-soluble and purified separately into three biologically active compounds on thin-layer chromatography. These compounds were purified (FIG. 2) and one of them was confirmed as gliotoxin (FIG. 1, n=2). Authentic gliotoxin was found to have similar anti-phagocytic and immunomodulating activity as the purified sample.
A method for the isolation of substantially pure gliotoxin from fungal cultures is provided by the following examples: